(326h) Chemoenzymatic Labeling of Recombinant Proteins Enables Rapid In-Lysate Bioconjugation and Assay Development

The selective conjugation of proteins to reactive partners is central to a diverse array of engineering applications, including the development of biosensors, self-assembled materials, and therapeutics. Therefore, methods that enable controlled, site-specific incorporation of bioorthogonal reactive groups into proteins are highly useful. Bioconjugation often requires extensive effort to first purify a protein of interest, and purification protocols may expose the protein to conditions detrimental to its structure and function. Thus, the ability to perform in situ bioconjugation in a protein’s native environment is advantageous. Here, we demonstrate co-translational protein labeling with an azide-bearing fatty acid by employing N-myristoyl transferase (NMT), a eukaryotic enzyme that appends myristic acid (Myr) to the N-terminus of its substrate proteins and that exhibits no activity toward bacterial proteins.

NMT-mediated protein labeling is quantitative and site-specific and can be engineered so that protein function is not inhibited by the addition of Myr-analogs. Here we report the labeling of proteins that are native NMT targets as well as proteins that are engineered to display NMT recognition sequences. In both instances, we find that addition of an azide-bearing fatty acid tag (12 azidododecanoic acid, 12-ADA) does not interfere with the proteins’ native function. Furthermore, we demonstrate bioconjugation of 12-ADA-labeled proteins to surfaces directly from soluble cell extracts via strain-promoted azide-alkyne cycloaddition. We find that the cycloaddition reaction rapidly and specifically conjugates 12-ADA-labeled proteins to microarrays in a concentration-dependent manner. Additionally, we conjugate 12-ADA labeled proteins to surface plasmon resonance (SPR) chips and measure the kinetics of binding of partner proteins. Of particular significance is the demonstrated ability to immobilize tagged proteins directly from cell lysate without prior protein purification, thus enabling the measurement of protein activity and binding dynamics with fast, reproducible assay platforms and significantly improving the throughput and quantitative characterization of protein function.